Hepatitis C virus (HCV) infection afflicts approximately 170 million people worldwide and chronic virus infection in these individuals is a significant cause of hepatocellular carcinoma (HCC). The mechanisms by which HCV induces HCC are largely unknown, but it is likely that virus induced alteration of host cell signaling pathways to create a replication niche for the virus and avoid the host anti-viral program is an important component of this pathogenesis. Using genomic scale siRNA screening, we have identified the host protein CARD14 as an important factor for HCV RNA replication. CARD14 is a known regulator of the NF-kB pathway, but its precise function in biology is unknown. Our preliminary data shows that infection of cells by HCV leads to a dramatic increase in NF-kB activation, and this activation is dependent on the presence of the CARD14 protein. Many infectious agents alter NF-kB gene expression to promote survival of the host cell, and the long-term manipulation of this pathway during chronic HCV infection may be one factor leading to the development of cancer. Our hypothesis is that CARD14 dependent activation of the NF-kB pathway is required for efficient HCV RNA replication and the prevention of apoptosis in infected cells, and that manipulation of this pathway by HCV is an important factor in the development of HCC. The details of how CARD14 functions in NF-kB signaling, how the virus manipulates this pathway, and what changes in gene expression result from this manipulation are important questions that need to be addressed, and we have designed four specific aims to address these important questions. We propose to validate the connection between CARD14 and NF-kB activation, map what regions of CARD14 are required for NF-kB activation and HCV replication, determine if CARD14 is functioning as a regulator of apoptosis, define proteins that are part of the CARD14 signaling pathway, and determine what cellular genes CARD14 manipulates during HCV infection. The completion of these aims will add considerably to our understanding of the cellular requirements for HCV replication, the manipulation of NF-kB signaling by HCV to promote cell survival, and the function of CARD14 in these processes. These experiments are of great potential significance to the development of HCC in those infected with HCV.